Novel biological substance from a fungus and the process for producing the same

ABSTRACT

This invention relates to a biological substance extracted from the Mycelium of a Fusarium. This substance has a peptidolic structure with specific amino-acids into. 
     This invention also relates to a process for producing the afore-said biological substance by culturing the strain of Fusarium and extracting the mycelium. 
     The biological substance has a therapeutical use.

PRIOR ART

The relevant prior art may be illustrated with the following references:

Belgian patent, 847 941

J of Antibioties, 29 (1976) 1043

J of Antibioties, 29 (1976) 1050

Tetrahedron, 14 (1978) 1147

SUMMARY OF THE INVENTION

This invention provides a biological substance obtained by extractingthe mycelium produced during the cultures of a Fusarium equiseti.

This biological substance contains four different components denominatedA, B, C and D of peptidolic structure. Only the specific amino-acidsvary from one component to another one.

This substance is produced by culturing a strain of Fusarium equisetiunder specific conditions, separating the mycelium and extracting itwith water unsoluble organic solvents. The biological substance isfurther purified according to the usual methods of the biochemistry.

This substance is of value for the immunological therapy and is used inthe form of pharmaceutical compositions, namely those suitable forparenteral, permucous, per cutaneous, sublingual or rectal ways.

PREFERRED EMBODIMENT OF THE INVENTION

This invention relates to a novel biological substance extracted from afungus.

More precisely, this invention provides a biological substance obtainedfrom the brothes of a Fusarium, a method for its production and its usein the form of pharmaceutical compositions.

This invention specifically provides a biological substance having apeptidolic structure obtained by extraction from the mycelium ofFusarium equiseti. This biological substance includes four differentcomponents having a very close structure and giving rise to theproduction by hydrolysis in acidic medium of a single hydroxy acid,α-hydroxy valeric acid and of specific α-amino acids i.e. N-methylValine, N-methyl isoleucine and N-methyl allo iso leucine.

The percentage of these four components named A, B, C, D has beendetermined after separation by high pressure liquid-chromatography inreverse phase, by means of an integrator. The percentage of fourcomponents in this biological substance is determined by measuring thesquare of each peak after high pressure liquid chromatography and theresidual absorption in UV light at 254 μm.

The respective percentages of each component may slightly vary and inaverage range between the following figures:

component A, 2-5%

component B, 10-16%

component C, 32-39%

component D, 40-47%

Component A after partial hydrolysis in alkaline medium provides 3 molof the lactone of α-hydroxy isovaleroyl N-methyl Valine. This lactoneresults from the splitting of the ester functions and recyclizing of theliberated hydroxy group into a lactone.

Component A after complete hydrolysis in acidic medium produces 3 molesof N-methyl Valine and 3 moles of α-hydroxy isovaleric acid. The acidhydrolysis produces both a split of the ester functions and of the amidfunctions.

Component B after partial hydrolysis in alkaline medium gives raise tothe production of 2 moles of the lactone of α-hydroxy isovaleroylN-methyl Valine and a mole of the lactone of α-hydroxy isovaleroylN-methyl isoleucine (or N-methyl isoalloleucine).

Component C under similar experimental conditions provides after partialhydrolysis 2 moles of the lactone of α-hydroxy iso valeroyl N-methylisoleucine (or N-methyl alloisoleucine) and 1 mole of the lactone ofα-hydroxy iso valeroyl N-methyl valine.

The complete hydrolysis in acidic medium gives rise to the production of3 moles of α-hydroxy isovaleric acid, 1 mole of N-methyl valine and 2moles of N-methyl isoleucine (or N-methyl alloisoleucine).

Compound D provides after partial hydrolysis in alkaline medium 3 molesof the lactone of α-hydroxy isovaleroyl N-methyl isoleucine (or N-methylalloisoleucine).

After complete hydrolysis in acidic medium, compound D provides 3 molesof α-hydroxy isovaleric acid and 3 moles of N-methyl iso leucine (orN-methyl alloisoleucine). In summary, the complete hydrolysis of thedepsipeptide according to the invention, provides α-hydroxy isovalericacid, N-methyl valine, N-methyl iso leucine (erythro form) and N-methylallo isoleucine (threo form). The N-methyl allo isoleucine has not yetbeen attributed to one specific component of the biological substance.

This biological substance of peptidolic structure may be obtained bysumitting to an aerobic fermentation a culture of Fusarium equiseti indetermined conditions of temperature, stirring and duration, separatingat the end of the time of fermentation the mycelium of Fusarium equisetifrom the broth, extracting the mycelium with a water-insoluble organicsolvent, separating the organic phases, evaporating off, taking up thecrude residue with a liquid hydrocarbon, submitting the organic solutionto a counter-current extraction with a mixture of water and a loweralkanol, separating this alkanolic phase, concentrating it andrecovering the biological substance which is further purified by theusual physical or chemical methods.

The process of preparation according to the invention may also bedefined by the following features, which are the presently preferredones:

the strain of Fusarium equiseti responsible of the production of thedepsipeptide according to the invention is that of Fusarium equiseti(Corda) saccardo, deposited at the Commonwealth Mycological Institute atKew (Surrey) Great Britain under the n 213 107.

the aerobic fermentation is carried out in three stages at a pH between5 and 6 and for a period of time extending from 36 to 48 hours for eachstage.

the extraction of the mycelium of Fusarium equiseti is performed using achlorinated organic solvent as for example Trichlorethylene.

the taking up of the crude residue is performed using a saturated liquidhydrocarbon such as hexane or pentane.

the purification of the peptidolic substance is performed by means ofchromatography on silica.

the hydrosoluble alkanol is methanol or ethanol.

The depsipeptide according to the invention is endowed with interestingpharmacological properties. It possesses strong immuno-modulatingproperties evidenced by a positive response in the test ofhypersensitivity to 4-ethoxy methylene 2-phenyloxazolone or tolipopolysaccharides of Escherichiacoli. Further the depsipeptide causesthe activation of macrophages by increasing or altering the enzymaticactivities and the total proteic content of the cells thereof. Moreover,the depsipeptide show an anti-exsudative and anti-oedematious activity.

Compared to the activities of Muramyldipeptide and of thelipopolysaccharides from Escherichia coli, the depsipeptides fromFusarium equiseti appears to be at least as active as these tworeference substances.

The depsipeptide according to this invention find a use in human orveterinary therapy, namely as a stimulant of the body defences. It isparticularly useful for the treatment of the chronic or acute diseasesof the respiratory tract such as chronic bronchitis and emphysema.

For the therapeutic use, the depsipeptide is incorporated intopharmaceutical compositions with an inert non-toxicpharmaceutically-acceptable carrier or vehicle. The carriers or vehiclesare those suitable for administration parenteral, permucous,percutaneous or rectal way.

As preferred pharmaceutical compositions they may be cited theinjectible solutions or suspensions, the sublingual tablets, thesolutions or suspensions in a diffusible solvent to be applied on themucosa optionally under pressure, the solutions in a polar solvent forthe percutaneous way and the suppositories.

The useful dosology may broadly vary depending of the therapeutic use,the way of administration, the weight or age of the patient. In the man,the usual dosology ranges from 0.05 and 1 mg per unit dosage and thedaily dosage ranges from 0.2 and 5 mg.

It may also be convenient to utilize discontinuous administrations suchas from 2 to 4 successive administrations followed with a stay forseveral weeks and renewed again 2 to 4 times.

The pharmaceutical compositions according to the invention may alsocontain further active ingredients having similar activities, orsynergistic activities; they also may contain diluing agents, fillingagents, binding agents, flavouring agents, sweetening agents,tensioactive agents, emulsifiers or suspending agents and propellingagents.

The pharmaceutical compositions are prepared according to the knownmethods of pharmacotechnology.

The following examples are merely intended to illustrate the invention:

EXAMPLE 1

Production of the depsipeptide from Fusarium equiset.

(A) Identication of the strain

The strain of Fusarium equiseti which allows the production of thedepspeptide has been identified as a Fusarium equiseti (Corda) Saccardoand deposited at the Commonwealth Mycological Institute at Kew under theNo. 213,107.

(a) fermentation brothes and mycelial growth

For the purpose of identifying this strain it appears convenient to usethe following methods of fermentation

(1) fermentation medium n°1

This medium is constituted from

    ______________________________________                                        saccharose              25g                                                   glucose                 25g                                                   ammonium nitrate        10g                                                   magnesium nitrate       2,5g                                                  mono potassium phosphate                                                                               5g                                                   water enough for        1000ml                                                ______________________________________                                    

This medium is sterilized and the pH value is adjusted aftersterilization to about 5.

After inoculation this medium gives raise to the production of a varydense mycelium, of a wooly appearance, and showing at its surface anorange colouration. At the reverse face in a Petri box thallus shows ayelllow-orange colouration, better known as a peach colouration turningto yellowish-orange in the aged cultures.

In the cultures on gelose it, appears a yellow pigment which diffusestherein.

(2) cultures on atomized corn steep (2%) agar-agar (2%) medium

In this medium the mycelium grows in producing a thallus appearing asrather close-shaved, wooly with concentric typical zonings. Pigmentationremains slight. The back free shows in the age a dark-yellowishcolouration.

(3) cultures on carboxy methyl cellulose

(Capellin+Petterson's Medium 1965) This medium is constituted with thefollowing ingredients:

    ______________________________________                                        carboxymethyl cellulose                                                                           15g                                                       ammonium nitrate    1g                                                        monopotassium phoshate                                                                            1g                                                        magnesium sulphate  5g                                                        yeast extract       1g                                                        ______________________________________                                    

Distilled water enough for 1000 ml

This medium may be optionaly added to small pieces of dry leaves ofPhragmites communes.

On this medium the mycelium remains scarce, very-close-shaved, nearlyunpercievable and colourless.

(b) effect of the light on the sporulation of the strain

Out the three above described medium under illumination, with whitelight during 12 hours per day only the medium containing carboxy methylcellulose and further the medium in which leaves of Phragmites communesare added, gives rise to the formation of macroconidias.

High amounts of macroconidias are formed in the culture media in Petribox enlighted with a Wood light diffuser (max. wawe lengths 360 and 365μm) disposed at 33 cm from the lamp, after an incubation period of 4 to6 days. The colouration of the media after culture under Wood light isslightly attenuated in comparison to that appearing in the culturesgrown uner white light.

(c) aspects of the Fusarium in several medias

Fusarium equiseti N° 213,107 has also been grown on usual media,generally utilized for the growth of various Fusarium. Experimentalresults have been thus obtained:

medium containing 40 g/l of flour of oat

medium containing 50 g of grated potatoes, 10 g glucose and 20 g gelosefor 1 liter water

medium containing 2% Agar and Malt

On these media, it appears that the conidiogenesis occurs with a veryslight extend with production of abnormalous conidias which could inducethe confusion between aborted macroconidias with microconidias.

It may be of interest to note some typical aspects of the Fusariumgenus, namely the appearance of a pigment distributed in concentriczonings. This coloured zones appear namely in the medium with potatoes,as well in the enlighted cultures as the cultures performed in the dark.

The cultures grown on the medium with flour of oat provide mycelialthalli which are short-shaved, compact, heterogenous, and slightlycoloured. Conidiogenesis did not practically occur.

MORPHOLOGICAL DATA OF FUSARIUM EQUISETI

Fusarium equiseti no. 213,107 shows the following particulars:

lack of microconidias

presence of macroconidias showing from 3 to 7 septa weared on branchedmacroconidiophors

presence of intermediate chlamydospori, isolate or in chain.

The whole morphological data of the macroconidias, the lack of terminalchlamydospori and the presence of intermediate chlamydospori allow theidentification of this Fusarium as a Fusarium of the subgenus Gibbosum.Among the three species included in this genus, only the speciesequiseti corresponds to this whole of morphological data. The appearanceof the peach colour of the cultures and the oblong shape of the basalcells in form of foot, confirm this identification.

(B) Preparation of the first tank of the fermentation (1) culture oninclinated gelose

The content of an ampul of lyophilizated mycelium is taken up with 2 mlsterile water. The thus obtained suspension is utilized for seeding 4test tubes containing 10 ml of a solid medium containing:

    ______________________________________                                        floaked oats             30g                                                  glucose                  10g                                                  agar-agar                20g                                                  distilled water enough for                                                                             1000ml                                               ______________________________________                                    

This solution is sterilized by heating for 20 mn at 120° C. The pH ofthis medium is about 6,6 before sterilization and 5,8 aftersterilization. These slides are incubated for 6 to 8 days in athermostatic oven at 28° C. then let to receive the sunlight for a weekat laboratory temperature.

(2) culture in a Roux's Flask

The aerial mycelium from a test tube is taken up with 10 ml sterilewater. This suspension is utilized for seeding 5 Roux's Flaskscontaining 200 ml of the same medium containing gelose as in the firststep. The incubation is carried out for 6 to 8 days in a thermostaticoven at 28° C. then subjected to enlightening in the sun light for aweek at the laboratory's temperature.

(C) Preparation of the inoculates for the second tank (a) first stage

From the culture in a Roux's flask, a suspension of mycelium in 100 mlwater is prepared by scraping the whole surface of the culture. Thissuspension is used for seeding a 10-liters Flask containing 4 liters ofthe following medium:

    ______________________________________                                        saccharose              25g                                                   glucose                 25g                                                   ammonium nitrate        10g                                                   mono potassic phosphate  5g                                                   magnesium sulphate      2,5g                                                  water enough for        1000ml                                                ______________________________________                                    

This solution is made sterile by heating it for 30 mn at 120°. The pHvalues decrease from 5,45 before sterilization to 4.75 aftersterilization. The aerobic incubation is performed at 28° C. in athermostatic jacket under bubbling of sterile air which insures thestirring for at least 48 h and better 72 hours.

(b) second stage

The seeding of the second tank is performed starting from the culture inthe 10 liters flask to a tank containing 75 liters of the followingmedium:

    ______________________________________                                        saccharose              40g                                                   celerose                 5g                                                   ammonium nitrate        10g                                                   monopotassium phosphate  5g                                                   magnesium sulphate      2,5g                                                  water enough for        1000ml                                                ______________________________________                                    

sterilization of this medium at 120° for 30 mn; pH after sterilization,5.6.

The fermentation of this second stage is performed under a stream ofsterile air of 3.5 m³ /h a stirring of 70 RPM, a temperature of 29° C.+1for at least 36 hours.

During the fermentation, the controls of sterility, the determination ofpH a nicroscopic evaluations and determination of the amounts ofreducing sugars are performed on samples sterilely taken up.

(c) third stage

A sample of the cultures of the second stage is used for seeding 12,001of the same milieu as previously (amount of culture: 6.25%).

    ______________________________________                                        aeration            70 m.sup.3 per hour                                       stirring            30 RPM                                                    temperature         29° C. + 1                                         duration from       36 to 40 h.                                               ______________________________________                                    

The broth is submitted during this period to the same determination aspreviously exposed.

(D) Industrial Manufacture

The 12,001 of inoculum (3rd stage) are utilized as a whole to seed anindustrial tank containing 7 m³ of a medium having the followingcomposition:

    ______________________________________                                        saccharose              50g                                                   Celerose                 5g                                                   ammonium nitrate        10g                                                   monopotassium phosphate  5g                                                   magnesium sulfate       2.5g                                                  zinc sulfate            .04g                                                  calcium carbonate       .2g                                                   tap water enough         1l                                                   ______________________________________                                    

This medium is sterilized by heating at 120° for 30 mn.

The pH of the medium after sterilization is about 6.5. This culture iscarried under the following experimental conditions.

    ______________________________________                                        aeration            250 m.sup.3 /h                                            stirring             20 RPM                                                   temperature          29° C. + 1                                        duration about       40 hours                                                 ______________________________________                                    

The pH value is of about 3 at the 24th hour and increases steadily toreach the values of about 5.3, 5.5 at the end of the fermentation. Aftertermination of the fermentation, the mycelium is separated from thebroth by filtration. The mycelium is dried in a ventilated oven 120 Kgmycelium are obtained after drying and grinding.

(E) Extraction of the depsipeptide (1) Extraction of the raw product

100 kg of the ground dried mycelium previously obtained are taken up in400 l trichlorethylene. After 2 hours stirring the unsoluble material isseparated by filtration and pressed. This mass is a new washed with 100l trichlorethylene then extracted a second time with 300 ltrichlorethylene under stirring for one hour and filtered. The insolublematters are further washed with 100 l trichlorethylene. The organicsolutions are united, concentrated under reduced pressure until a thickmass is obtained. This mass is separated and weighs about 10 Kg.

It is further taken up in 90 liters hexane. The solution is filtered andthe clear filtrate is extracted using a counter current device withaqueous methanol (methanol 80: water 20 vv). Once with 50 l, once with25 l, for the third time with 12.5 l and for the fourth time with 10 lof the mixture. The methanolic solutions are united and distilled offuntil dry under reduced pressure.

(2) Purification of the raw product

1 Kg of alumina is suspended in 5 l water and concentrated hydrochloricacid is slowly added until the pH value reaches 4. The stirring is keptfor 24 h while maintaining the pH at this value. Alumina is filtered ona gauze of nylon, washed with water then with acetone until dry andfinally dried in an oven at 200° for a night.

20 Kg of the methanolic dry residue are dissolved in 80 liters methylenechloride and the solution is filtered. The filtrate is poored on achromatography column filled with 40 Kg of alumina prepared aspreviously indicated and further washed with methylene chloride. Themethylenic solution is slowly added at a speed of about 15 liters perhour. After completion of the addition the column is washed at the samerate with 80 liters of methylene chloride.

The methylenic eluates are recovered and concentrated until thickconsistence. This paste is taken up in 200 liters ethanol, filtereduntil clear, cooled to 4° C. and slowly added to 400 liters distilledwater. The addition of water lasts about 8 hours and the thus formedsuspension is kept under stirring for 20 hours at 4° C. The precipitatedcrystalls are separated by filtration, washed with 20 l of a mixture ofethanol: 1-water: 2 previously cooled at 4° then with 50 l water. Thecrystalls are further dried at 40° C. in an oven under reduced pressureuntil constant weight.

The depsipeptide appears as a white crystalline powder, odourless and ofbitter taste. It is sparingly soluble in water but easily soluble inchloroform, methanol and 95% ethanol.

The melting point of the anhydrous product determined on Koffler bank isabout 121° (+5); Its rotatory power is [α]_(D) ²⁰ =-80° (+5) (C=5%methanol)

Nitrogen content: 6,2+0.3%

The infra-red spectrum in KBr is supplied with hereafter.

(3) Separation and identification of the four components by TLC

A solution of 10 mg/ml of the depsipeptide in methanol is prepared and10 μl of this solution is deposited on a plate of 20 cm×20 cm chargedwith a mixture of cellulose Mn 300 and cellulose Mn 300 Q andimpregnated with a mixture of formamide-acetone (1:4) until 2 cm fromthe top.

After development the plates are dried at 100° C. for 1 hour, andsprayed with a 0.5% chloroformic solution of iodine. The plates show 4different yellow strains. The two most important have a Rf respectivelyof 0.55 and 0.45, one less important has a Rf of about 0.35 and theslighest one has a Rf of about 0.3.

(4) Separation of the components by HPLC

The percentages of the four components after HPLC have been determinedwith an integrator by reference to the square of the 4 peaks andassuming that the absorption in UV light at 254 mμ is the same for eachcomponent.

The HPLC in reverse phase has been carried out with a 30 cm long columnfilled with Bonsapak CL 8 ml in a Waters apparatus. The solvent ofelution is a mixture of methanol 8V-water αv. The output of the eluentsis of about 1.5 ml/mn and 10 μl of a 10 mg/ml solution of thedepsipeptide in methanol is injected. The peaks are detected by measureof the absorbance at 254 mμ, and the squares thereof is measured with anintegrator ICAP 5.

The results obtained correspond to the average of the values obtainedwith several samples of different productions of the depsipeptide.

    ______________________________________                                        Compound A in average                                                                           3.9                                                         Compound B in average                                                                           15.3                                                        Compound C in average                                                                           38.1                                                        Compound D in average                                                                           42.7%                                                       ______________________________________                                    

The following graph shows the separation of the four components by HPLC

EXAMPLE II

Pharmaceutical compositions containing as active ingredient thedepsipeptide from Fusarium equiseti.

(a) pressurized solution in Isopropyl Myristate

    ______________________________________                                        Depsipeptide             .050g                                                Oil of Neroli            .050g                                                Eucalyptol               .050g                                                Saccharine               .00125g                                              Isopropyl Myristate enough for                                                                        2.5ml                                                 Flugene 12 as propellent                                                                              7.5ml                                                 ______________________________________                                    

(b) aqueous suspension

    ______________________________________                                        Depsipeptide               .050g                                              polyoxyethylenesorbitane mono oleate                                          sold under the trade name "Tween 80"                                                                     .010g                                              Sodium chloride            .180g                                              Oil of Neroli              .050g                                              Eucalyptol                 .050g                                              Sodium saccharinate        .010g                                              Water enough for           20ml                                               ______________________________________                                    

The two pharmaceutical compositions are packed in a flask fitted with apump which delivers each time 0.080 g of solution or suspension i.e. 200μg of active ingredient by delivered dosis.

(c) pressurized solution in Isopropyl Myristate

    ______________________________________                                        Depsipeptide             .015g                                                Flavouring agent         .0075g                                               Isopropyl Myristate enough for                                                                         .75ml                                                Freon 12                2.25ml                                                ______________________________________                                    

This solution is packed in a container fitted with a valvedelivering 25μl each time. The amount of depsipeptide delivered at each pulverizationis of about 125 μg.

(d) pressurized solution in Isopropyl Myristate

    ______________________________________                                        Depsipeptide            .030g                                                 Flavouring agent        .015g                                                 Isopropyl Myristate     .75ml                                                 Freon 12               2.25ml                                                 ______________________________________                                    

This solution is packed in a container fitted with a valve delivering250 μg of depsipeptide each pulverization. This container is intendedfor 120 pulverizations.

(e) Solution in oil

    ______________________________________                                        Depsipeptide            .05g                                                  Saccharine              .0015g                                                Flavouring agent        .05g                                                  Isopropyl Myristate enough for                                                                        6ml                                                   Propelling agent        .05g 2ml                                              ______________________________________                                    

The container is fitted with a valve delivering 30 μl each time.

(f) aqueous suspension

    ______________________________________                                        Depsipeptide               .050g                                              polyoxyethylenesorbitane mono oleate                                          (sold under the trade name "Tween 80")                                                                 120mg                                                Potassium chloride        3mg                                                 Disodium phosphate        17.25mg                                             Dipotassium phosphate     3mg                                                 Calcium chloride          1.5mg                                               Magnesium chloride        1.5mg                                               Sodium Mercurithiolate     .0375mg                                            Water enough for          15ml                                                Propellent enough                                                             ______________________________________                                    

This suspension is packed in a container fitted with a pump delivering75 μl at each pulverization.

Each pulverization then provides 250 μg of active ingredient.

(g) injectible aqueous suspension

    ______________________________________                                        Depsipeptide            100mg                                                 Cremophor EL            100mg                                                 Sodium chloride         800mg                                                 Sterile water enough for                                                                              100ml                                                 ______________________________________                                    

This suspension is packed in ampuls of 1 ml each containing 1 mg activeingredient per unit dosage.

EXAMPLE III

Pharmacological study of the depsipeptide of this invention

(A) Study of the stimulation of the humoral immunity in the mice aftersensibilization with red blood cells of the sheep

This test is performed on batches of 10 mice of swiss CD strain weighingabout 20 g. Each mouse receives a threshold immunizing dosis of 10millions of red blood cells by intravenous way.

The mice are treated at various periods of time before immunizationeither by intravenous or intraperitoneous way. The depsipeptide fromFusarium equiseti is dissolved in propylene glycol (84 mg/ml) then thisconcentrated solution is diluted with a saline solution. The mice areinjected with 10 or 50 μg of depsipeptide.

A lot of controls receives only the red blood cells and the solvent.

A lot of mice receives a suspension of a culture of Corynebacteriumsimplex in a saline solution as a reference product.

The bloods of the mice are taken at the established days throughretroorbital ponction. The sera are stored at -80° C. For each batch thesera are tested individually as well as a pool of sera.

DETERMINATION OF THE CONTENT IN ANTIBODIES (a) determination of thehemagglutinine titer

0.05 ml of a suspension of red blood cells of sheep in the Mayer buffer,titrating 7.107 cells/ml is added to 0.025 ml of serum diluted in asaline s solution. The plates are placed for two hours at roomtemperature in a closed vessel then for a night at +4°. Thehemagglutinations are evidenced by reading through a magnifier lens. Thehemagglutinizing titer is calculated from the highest dilution of theserum providing a clear hemagglutination. It is expressed using theexponent at the log 2 of this dilution (for example dilution 1/64titer=6)

The clear hemagglutinations are only shown from titer=4, the lowestvalues being usually not clearly concluding.

(b) determination of the hemolytic titer

0.025 ml of a suspension of red blood cells of sheep in the Mayer'sbuffer (10⁸ /ml) are added to 0.025 ml of guinea pig's complementpreviously diluted in the Mayer's buffer (1/200) and to 0.025 ml serumdiluted with saline solution (1/2, 1/4, 1/8 . . . )

The plates are placed in an oven at 37° for 1 hour then at +4° C. for 2hours. The determination is effected by means of a glass.

The hemolytic titer is appreciated as the highest dilution of the serumcausing the complete lysis of the red blood cells of sheep. It isexpressed in the same fashion as the hemagglutining titer.

RESULTS (1) Study on the threshold dosis

Several dosis of red blood cells of sheep have been tested in order todetermine the minimal dosis which causes an humoral answer in the swissmice and provokes a supraliminal answer. The maximal answer obtainedwith 10⁸ red blood cells/mouse is magnified by the immunostimulantagents. Moreover 10⁶ red blood cells do not provoke any sensible humoralanswer. In contrast thereof 10⁷ red blood cells/mouse give a homoralanswer the cinetic of which is of value for the testing of theimmunostimulant agents.

The threshold dosis may vary broadly without any scientific explanation.

(2) Study of the kinetics in the controls and in reference animals

Several batches of controls which receive only 10⁷ red blood cells/mousehave been studied from a point of view of kinetics and have shown thatthe hemagglutinizing antibodies appear from the fourth day afterimmunization for certain batches and from the seventh day for otherbatches. The hemagglutining titers are usual lower than 4. It appearsalso that the hemolytic titers of the sera of the controls arepractically equal to zero until the seventh day.

Cultures of corynebacterium parvum have been used as a referencesubstance and show they stimulate the immunological answer beinginjected 3 to 6 days before the injection of red blood cells. Antibodiesappear in immunostimulated animals on the fourth day. 14 days after thesignificance of the immunological answer is statistically of greatervalue.

(3) Study of the depsipeptide from Fusarium equiseti

The depsipeptide from Fusarium equiseti has been administered to miceduring 3 days from the 6th to the third day before injection of redblood cells. It provokes an increase in the content of hemolytic andhemagglutining antibodies together with an induction in the appearanceof hemolytic antibodies from the fourth day after injection of red bloodcells.

Doses from 10 μg to 50 μg/day/mouse cause the same result. Theimmunostimulation caused by the depsipeptide is of the same order ofthat caused with 500 μg/day of cultures of corynebacterium parvum(Institute Pasteur of Paris). The same results have been obtained withthe depsipeptide with doses which are 10 to 50 times lower than those ofcorynebacterium parvum.

(B) Lymphoblastic transformation Test (TTL)

The determination of the lymphoblasts by administration of thedepsipeptide from Fusarium equiseti has been carried out using themethod described by R. P. Danicle and S. K. Hollan Proceed Nat. Acad Sci(New York) 73, (1976) 3599. This test is performed at the optimalconcentration of the mitogenic agent, the product to be tested remainingin contact with the cells during the whole set of time. Under thisexperimental conditions the, incorporation of the biological precursoris quite reproductible and maximal. The biological effect produced bythe product is evidenced by inhibition of the incorporation of thetagged inhibitor.

The determination of the optimal doses of the mitogenic agent is basedon the curves Effect/Doses using the beginning of the plateau.

The reference substances are Phytohemagglutinin (optimal dosis 1 μg/ml)and lipopolysaccharides of Escherichia coli (optimal dosis 5 μg/ml)

From the results hereafter exposed, it appears that in contrast to thereference substances the depsipeptide from Fusarium equiseti is not amitogenic agent at the tested doses and curbes the incorporation oftritiated Thymidine usually induced by the mitogenic agents.

(C) Study of the effects of the depsipeptide from Fusarium equiseti onthe immunological response

Depending on the administered doses the tested substance show astimulant or depressant effect either on the humoral or cellularimmunological answer in the mice.

The humoral answer has been evidenced after administration oflipopolysaccharide from Escherichia coli (Vujanovic 1973-Le Bouteiller1974) which increase the amount of blood cells of medullar aspect.

The cellular response has been evidenced after administration ofethoxymethylene oxazolone according to the methods described by Turk(1967) and Anderson (1972).

The characteristics of the cells from an immunological andultrastructural point of view have been determined according to themethod of Jeannesson (1975)

The number of white blood cells producing the antibodies isquantitatively determined using the test of hemolysis zones in a semisolid medium containing the target red blood cells. This test utilizesthe zones of direct hemolysis according to the method of Jerne andNordin (1963) which detect the cells producing Ig M

In view of the auto-radiographie study, the suspensions of cells areincubated at 37° C. with tritiated thymidine at a dosis of 20 μCi/ml for30 minutes before being treated for the determination of the Ig M ofsurface.

The cells are fixed with glutaraldehyde then stained withdiaminobenzidine according to the method of Graham and Karnovsky (1966),then treated with osmic acid and inbedded into EPON.

RESULTS Immunological response to ethoxymethylene oxazolone (a) numberof cells

On the 5th day after administration of the depsipeptide from Fusariumequi seti the increase of the number of cells is usually higher than 50percent, namely after intraperitoneal administration in the mice. On the9th day the increase is slightly less significant and thereafterdiscontinues. The number of cells having incorporated some tritiatedthymidine is of the same order by controls and tested animals, i.e. ofabout 4-6%.

(b) number of cells able to recognize the antigenic substances

On the 5th day the percentage of cells recognizing the antigenicsubstance is very significantly increased (257 percent) and thisincrease results both from an increase of the amounts of cells in theganglions and an increase of the percentage of the cells specificallyactivated for a determined number of cells (10⁶ cells). This increaseprogressively disappears and cannot be evidenced after the 9th day.

(c) number of cells forming zones of hemolysis

The very significant increase of cells forming zones of hemolysisappears on the 5th day and is caused by an increase in the number of thecells. On the 9th day this increase namely results from the number ofcells forming hemolysis zones for a determined number of cells (10⁶cells).

(d) effect on the content in antibodies

On the 5th day the content in antibodies remain constant from thebeginning. On the 9th day the percentages of hemagglutining antibodiesand of hemolyzing antibodies is broadly increased namely that ofhemolyzing antibodies.

(e) study on the number of cells having Ig on their surfaces

The cells having Immunoglobulines (Ig) at their surface have beencounted on their slides from the initial cellular suspension in thecontrols and in the treated mice. The percentage is of about 35 percent.

From a microscopic inspection it appears that the nature of the cellscausing hemolysis and the cells forming rosettes are the same in thecontrols and in the treated mice. For the rosette forming cells they arelymphokystes with Ig on their surface and white T cells the proportionof which reaches 40 to 50 percent on the 9th day. For the hemolysiszones forming cells they are namely plasmocyts.

IMMUNOLOGICAL RESPONSE AFTER PRETREATMENT WITH LIPOPOLYSACCHARIDES

The injection of the depsipeptide from Fusarium equiseti induces aslight increase in the number of cells on the 9th day.

The effect on cells recognizing the antigenic substance is none and evennegative. The decrease of the cells able to recognize the antigenicsubstance is more significative on and after the 9th day.

No effect has been evidenced on the number of cells forming hemolysiszones.

A slight decrease in the content of hemagglutining antibodies appearswhilst an important increase in the content of hemolyzing antibodies isshown on the 5 th day.

CONCLUSION

The immunological effects of the depsipeptide from Fusarium equiseti maybe summarized in the following fashion.

Immunological response to ethoxymethylene oxazolone is increased. Theincrease of the cells in or around the ganglions is significant andmaximal on the 5th day (50 to 80%). The number of cells recognizing theantigenic substance is very frequently increased either due to theincrease of the number of cells or due to the increase in this specificactivity. The increase in the hemolysis zones forming cells reflectsonly the increase in the number of cells in the ganglions.

The immunological response after pretreatment with lipopolysaccharidesis decreased. The number of cells recognizing the immunogenic agentremains constant or is decreased. The content in antibodies is decreasedor remain unaltered.

(D) Effect of the depsipeptide on the immunological response. (1)Potentializing action on the humoral immunity.

This test has been performed on batches of 10 mice (C 57 B6 male micestrain) which are injected each with 300 μg of purified bovine serumalbumine (fraction V of Cohn), purchased from the Armour PharmaceuticalCompany, in the posterior plantar paws.

At the first day 20 mice received each 0.2 ml of a mixture containingthe solution of BSA in a phosphate buffer and 10 μg of the depsipeptidefrom Fusarium equiseti (2 vol) and Freund's uncomplete adjuvant (3 vol).

A batch of 10 mice is used as controls. It receives only the samemixture but the depsipeptide and the Freund's adjuvant.

Anoter batch receives only the injection of BSA together with theFreund's adjuvant.

At the day 2I the half of the mice in each bath is sacrifized todetermine the content of anti BSA-antibodies by passivehemagglutination. The second half of the mice receives a reniewedinjection of the mixture at half dosage (0.1 ml instead of 0.2 ml).

At the day 35 the remaining mice are sacrifized and the content ofspecific anti BSA antibodies is determined using the same method.

A very significant increase in the content of specific antibodies isevidenced on the 35th day in the treated mice.

(2) Effects on the cellular immunological response. (a) mitogenicpotency of the depsipeptide

500.000 splenic lymphokytes from CBA strain mice suspended in 0.1 mlRPMI are incubated at 37° C. for 3 days in a stream of carbonicanhydride with various concentrations of the depsipeptide from Fusariumequiseti.

In the same fashion the white cells are also incubated withphytohemagglutinine at a concentration of 1/200, Concanavaline at aconcentration of 10 μg/ml as reference substances.

4 hours before the termination of this incubation period, 1 μCitritiated thymidine is added to each culture. Afer completion of theincubation the cells are filtered and twice washed with saline solution.The radio activity on the filter is counted with a β-ray counter. Theincorporation of Thymidine is calcuated as the ratio between the amountof incorporated thymidine in the presence of the mitogenic substance andthe amount of incorporated thymidine in the lymphokytes without anymitogenic substance.

RESULTS

The following results have been obtained and are tabulated hereinafter:

The results show that the depsipeptide curbs the nucleic metabolism atvery low dosages through a very strong effect on the immunosuppressivecells or a modification of the metabolism or cellular permeability.

The depsipeptide is not a mitogenic substance as such. In contrastthereof, it may have a stimulant effect on the lymphokytes in thepresence of the usual mitogenic substances.

    ______________________________________                                        Amount of  Ratio of                                                           active substance                                                                         incorporation                                                                             Statystical value                                      ______________________________________                                         0,1  pg/ml =  0,41 (± 0,1)                                                                           p = 0,0047                                          1    pg/ml =  0,62 (± 0,07)                                                                          p = 0,044                                           5    pg/ml =  0,84 (± 0,06)                                                                          Not significative                                   10   pg/ml =  0,90 (± 0,06)                                                                          Not significative                                  110   pg/ml =  0,32 (± 0,12)                                                                          p = 0,0078                                          1    ng/ml =  0,40 (± 0,10)                                                                          p = 0,0082                                          10   ng/ml =  0,56 (± 0,08)                                                                          Not significative                                  100   ng/ml =  0,62 (± 0,07)                                                                          Not significative p = 0,054                         1    μg/ml =                                                                             0,36 (± 0,1)                                                                           p = 0,0044                                          10   μg/ml =                                                                             0,63 (± 0,07)                                                                          p = 0,028                                          100   μg/ml =                                                                             0,80 (± 0,06)                                                                          Not significative                                  PHA   1/200 =  51,15 (± 0,006)                                                                        p = 0,0028                                         ConA  10 μg =                                                                             50,30 (± 0,006)                                                                        p = 0,0025                                         LPS   100 μg =                                                                            14,81 (± 0,012)                                                                        p = 0,0028                                         ______________________________________                                    

(b) stimulation of the activities of the macrophages

The peritoneal macrophages from unstimulated rats (Fisher strain) areprelevated through peritoneal washing and isolation on plastic rack.They are cultivated for a night with dosages of the depsipeptide rangingfrom 1 picogramm to 1 nanogramm/ml.

The content in proteins, in lysosomal β-glucoronidase, and incytoplasmic leucine-amino peptidase are determined on the cellspreviously lysed with 0.05% Triton X-100.

In the same manner, incubation has also been performed with a solutionof lipopolysaccharide from Escherichia coli and Muramyldipeptide asreference substances.

The depsipeptide induces a significant increase of the hydrolases in themacrophages at a dose ranging from 1 pg to 10 ng/ml. The proteic contentis also increased with doses ranging from 10 to 100 pg/ml. As higherdoses and namely from 100 ng/ml the depsipeptide appears to be toxic forthe cells as evidenced by the strong decrease in the proteic content andthe increase in the content of intracellular macrophagic enzymaticactivities.

The same test has also been performed in vitro. The rats received 6 daysbefore the peritoneal washings an intrapeitoneal injection ofdepsipeptide (1 to 100 μg in 1 ml saline solution).

Lipopolysaccharides from Escherichia coli Muramyl dipeptide and watersoluble adjuvant are injected to other batches of rats as referencesubstances. The peritoneal macrophages recovered after washings areincubated for 2 hours in order to delete the non-adhering cells andfurther for 4 hours. The cells are then lysed and the enzymatic andproteic contents determined as above given.

RESULTS

At a dosis of 1 μg the depsipeptide from Fusarium equiseti induces avery high increase of the lysomal enzymes (β-glucuronidase+35%) and ofthe cytoplasmic enzymes (leucine amino peptidase+35%). The content inproteins is also increased (+24%). At this dosis the water solubleadjuvant induces an increase of the hydrolases of 39% and thelipopolysaccharides an increase of 42% of the leucine aminopeptidase and35% of the proteins. These activities are thus of about the same level.

In contrast thereof at a dosis of 100 μg the depsipeptide induces anincrease of 32% of the protein content and of 50% of the cytoplasmicenzymes. These activities are far higher than those of the otherimmunostimulating agents.

At a dosis of 1 ng the stimulation is weaker.

protein content, +11%

β-glucouronidase, +23%

leucine amino peptidase, +16%

The following tables summarized the various results obtained withseveral doses of the depsipeptide in comparison with the referencesubstances.

                                      TABLE I                                     __________________________________________________________________________                      1 μg                                                                              P   100 μg                                                                           P                                          __________________________________________________________________________    Depsipeptide                                                                           Proteins + 24%                                                                              = 0,0125                                                                            + 32% 0,005                                               β-Glucuronidase                                                                   + 35%  0,025                                                                             + 37% 0,025                                               L.A.P.   + 35,5%                                                                              0,025                                                                             + 59% 0,0025                                              (leucine amino-                                                               peptidase)                                                           L.P.S.   Proteins + 35%  0,01                                                                              + 14% 0,025                                               β-Glucuronidase                                                                   - 14%  0,025                                                                             - 3%  N.S.                                                L.A.P.   + 42%  0,001                                                                             + 2%  N.S.                                       M.D.P.   Proteins + 7,5% N.S.                                                                              + 2%  N.S.                                                β-Glucuronidase                                                                   + 27%  0,01                                                                              + 5%  N.S.                                                L.A.P.   + 24%  0,025                                                                             + 5%  N.S.                                       W.S.A.   Proteins + 3,6% N.S.                                                                              + 1%  N.S.                                       (Water soluble                                                                         β-Glucuronidase                                                                   + 39%  0,001                                                                             + 46%                                                                             = 0,001                                      adjuvant)                                                                              L.A.P.   - 10,5%                                                                            = 0,025                                                                             - 7%  N.S.                                       __________________________________________________________________________

Amounts of cellular enzymes and proteins in the macrophages after 16hours incubation.

                                      TABLE II                                    __________________________________________________________________________                           Lipopoly-                                                                             Muramyl-                                                      Depsipeptide                                                                          saccharide                                                                            dipeptide                                      __________________________________________________________________________    Saline                                                                              Proteins 45.7 ± 0,1                                                                         46.0 ± 1,1                                                                         46,0 ± 1,1                                        β-glucuronidase                                                                   29,5 ± 0,1                                                                         29,1 ± 2,2                                                                         29,1 ± 2,2                                   1 pg/ml                                                                            Proteins 44,9 ± 4,2 NS                                                                      55,1 ± 2,2.sup.b                                                                   46,1 ± 2,2 NS                                     β-glucuronidase                                                                   35,8 ± 1,9.sup.c                                                                   39,2 ± 1,5.sup.b                                                                   36,5 ± 0,1.sup.b                             10 pg/ml                                                                           Proteins 53,3 ± 4,3.sup.b                                                                   47,7 ± 2,2 NS                                                                      44,8 ± 0,7 NS                                     β-glucuronidase                                                                   35,4 ± 4,8.sup.c                                                                   37,1 ± 5,8.sup.a                                                                   36,3 ± 6,4 NS                               100 pg/ml                                                                           Proteins 50,1 ± 4,2.sup.b                                                                   52,2 ± 6,3 NS                                                                      44,6 ± 1,9 NS                                     β-glucuronidase                                                                   33,0 ± 2,4.sup.c                                                                   38,7 ± 9,9 NS                                                                      41,0 ± 2,9.sup.b                             1 ng Proteins 48,2 ± 4,3 NS                                                                      45,0 ± 0,4 NS                                                                      44,0 ± 1,8 NS                                     β-glucuronidase                                                                   34,2 ± 3,7.sup.c                                                                   21,0 ± 0,5.sup.a                                                                   34,0 ± 5,3 NS                                10 ng/ml                                                                           Proteins 45,9 ±  1,4 NS                                                                     47,1 ± 0,4 NS                                                                      43,4 ± 1,8 NS                                     β-glucuronidase                                                                   32,8 ± 5,6.sup.b                                                                   25,2 ± 6,6 NS                                                                      39,6 ± 6,1.sup.a                            100 ng/ml                                                                           Proteins 44,3 ± 4,0 NS                                                                      52,3 ± 0,4.sup.b                                                                   40,5 ± 0,7.sup.b                                  β-glucuronidase                                                                   29,8 ± 8,8 NS                                                                      34,9 ± 3,4.sup.a                                                                   23,9 ± 6,2 NS                                1 ng/ml                                                                            Proteins 44,2 ± 5,9 NS                                                                      46,5 ± 1,3 NS                                                                      45,1 ± 0,9 NS                                     β-glucuronidase                                                                   27,1 ± 0,3.sup.c                                                                   26,0 ± 2,1 NS                                                                      30,6 ± 3,8 NS                                10 ng/ml                                                                           Proteins 41,9 ± 7,0.sup.b                                                                   43,7 ± 2,5 NS                                                                      48,1 ± 1,8 NS                                     β-glucuronidase                                                                   25,2 ± 9,3.sup.b                                                                   23,4 ± 1,7.sup.c                                                                   32,2 ± 3,0 NS                               100 ng/ml                                                                           Proteins 36,6 ± 5,6.sup.c                                                                   41,2 ± 3,8.sup.b                                                                   55,3 ± 3,1.sup.b                                  β-glucuronidase                                                                    7,5 ± 3,5.sup.c                                                                   11,6 ± 2,4.sup.c                                                                   39,0 ± 1,6.sup.c                            __________________________________________________________________________

The content in proteins is expressed in g/10⁶ cells and that inβ-glucuronidase in mMol of substrate hydrolysed by 10⁶ cells per hour.

Degrees of statistical significance

    ______________________________________                                        a          =            p 0;05                                                b          =            p 0,025                                               c          =            p 0,001                                               NS         =            without signification                                 ______________________________________                                    

(3) Effect on the release of chromium induced in the larvae ofschistosoma mansoni after treatment by an immunostimulant agent.

The depsipeptide from Fusarium equiseti has been tested to determine itsaction in vivo on the cytotoxicity induced in the intraperitonealmacrophages of the rat against the larvae of schistosoma mansoni.

The macrophages are stimulated either in vitro or in vivo and arecontacted with a suspension of larvae of schistosoma mansoni previouslytagged with 51 chromium.

Cytotoxicity in vivo

This contact is intended to demonstrate an optional cytotoxic effectmeasured by the amounts of chromium released in the culture medium. Thismeasure reflects the significance of cellular lesions on the surface ofthe schistosomules. The radio activity released in the medium isexpressed as a percentage of the total radio-activity present in theschistosomules at the beginning of the experiments.

The obtained results are summarized in the table III

    ______________________________________                                        Substances  1 ng       1 g        100 g                                       ______________________________________                                        Depsipeptide                                                                              +0,5% (NS) +13% (NS)  +10% (NS)                                   Lipopolysaccharides    +24%       +50%                                                               (p 0.025)  (p 0.005)                                   Muramyldipeptide       +6% (NS)   +10% (NS)                                   WSA                    +23%       +30%                                                               (p 0.025)  (p 0.025)                                   ______________________________________                                    

The despsipeptide causes a release of 51 Cr but it is not statisticallysignificant; it is also for Muramyldipeptide the same.

The lipopolysaccharides and the WSA make the macrophages significantlymore toxic for the larvae as are the macrophages of untreated animals.

The depsipeptide is practically devoid of any effect on the macrophagesin vivo against the schistosomules.

CYTOTOXICITY IN VITRO

After incubation for 24 hours of normal macrophages it is not anyincrease of the cytotoxicity of the macrophages against the taggedschistosomules. The doses of depsipeptide which stimulate themacrophages induce only a slight decrease of the release of the taggedtracer.

The table IV explained the obtained results

    ______________________________________                                        Release of 51 Cr                                                              Saline  100 pg/ml 1 ng/ml   10 ng/ml                                                                              100 ng/ml                                 ______________________________________                                        24.03%  19.67%    18.21%    21.71%  23.46                                     (± 2.4)                                                                            ± 3.1  ± 3.9  ± 3.5                                                                              ± 2.7                                  ______________________________________                                    

(4) Action of the depsipeptide on the survival time of the mice afterinjection of leukemic cells L 1210

Due to the fact that the depsipeptide from Fusarium equiseti acts as animmunostimulant in vivo and acts namely as activating the macrophages(as does BCG), it appears of value to study comparatively these twosubstances.

As a model they are utilized batches of mice inoculated with leukemiccells L 1210 and previously treated with Cyclophosphamide. The injectionof the depsipeptide or of BCG allows the determination of their actionon the residual action disease.

METHOD

This test has been performed in batches of mice (strain BDF) of 7-8weeks old. The animals are treated according to the method described byMathe and Cowork "Immunotherapie active des cancers" (ESP Paris 1976)

At the day 0 all the animals receive intravenously a suspension of 1000leukemic living cells L 1210.

At the day 1 the animals are divided in 4 lots of 10 animals of the sameweight. The controls receive only the saline solution by intraperitonealway. The three other batches receive intraperitoneously an injection ofcyclophosphamide at the suboptimal dosis of 80 mg/Kg. The batches whichreceive the cyclophosphamide are further treated in the followingmanner:

one batche receives intravenously 0.1 ml of saline solution at the day+6. This batch is merely intended to determine the time of survival ofthe mice previously treated by the cyclophosphamide.

one batch receives intravenously an injection of 1 mg/mouse of BCG(Institut Pasteur) in 0.1 ml saline solution.

They are intended to show the effects of the active immunotherapy afteradministration of cyclophosphamide, BCG appearing as an active agent inthese experimental conditions.

another batch receives intraperitoneously 50 μg Depsispeptide/mouse in0.5 ml saline solution. They are intended to determine the survival timeof the mice treated by the depsipeptide from Fusarium equiseti.

For each batch the survey of the mice is maintained for 40 days. Therole of each product on the survival time is estimated and thestatistical significance is determined using the test "t" of student bycomparing the results of each batch to those of the controls and ifnecessary with the results obtained with the treated batches.

RESULTS

The results obtained in this test are gathered in table V.Cyclophosphamide at suboptimal dosis extends in a statiscallysignificative manner, the survival time of the animals. The mixturecyclophosphamide-BCG merely improves the survival time in a nonstatistically significative manner.

The mixture cyclophosphamide-depsipeptide provokes a statisticallysignificative improvement of the survival time in the thus treatedanimals. However no significative difference can be seen between theresults from the two batches treated with the mixture of therapeuticagents (cyclophosphamide+BCG and cyclophosphamide+depsipeptide)

This test shows that the depsipeptide from Fusarium equiseti increasesthe survival time of the mice previously injected with 1000 L1210 livingleukemic cells and treated with cyclophosphamide. The results are of thesame order than those obtained with BCG the number of animals used inthis experiment is to small to establish a statistically significativedifferentiation.

Moreover it has been determined in the mice that a single injection ofthe depsipeptide from Fusarium equiseti (50 to 250 μg) induces theappearance of Hassal's corpuscules in the thymus and the appearance ofthe germinative center in the spleen.

DETERMINATION OF THE ACUTE TOXICITY

The average lethal dosis of the depsipeptide has been determined onbatches of 10 heteroxenic mice (strain CD) or on batches of 10 rats(strain Wistar). All the animals receive the depsipeptide either inaqueous suspension or in a solution into Isopropyl Myristate.

This compound is administered orally, intraperitoneously orintravenously at increasing dosages. The animals are kept under surveyfor 8 days, and the deathes when they are any, are numbered. The averagelethal dosis is graphically determined using the method of Lichtfieldand Wilcoxon.

The thus obtained results are hereinafter summarized:

(a) aqueous suspension

    ______________________________________                                        in the mice orally                                                                              LD.sub.50 = 5000 mg/Kg                                       intraperitoneously                                                                             LD.sub.50 = 67 mg/Kg                                         intravenously    LD.sub.50 = 17 mg/Kg                                        in the rats orally                                                                              LD.sub.50 = 5000 mg/Kg                                       Intraperitoneously                                                                             LD.sub.50 = 67 mg/Kg                                         intravenously    LD.sub.50 = 11 mg/Kg                                        ______________________________________                                    

(b) solutions in Isopropyl Myristate

    ______________________________________                                        in the mice intraperitoneously                                                 male mice         LD.sub.50 = 169 mg/kg                                       female mice       LD.sub.50 = 182 mg/Kg                                      in the mice intravenously                                                      male mice         LD.sub.50 = 41 mg/Kg                                        female mice       LD.sub.50 = 41 mg/Kg                                       ______________________________________                                    

The phenomena of toxicity after intraperitoneal or intravenous injectionare namely due to the physical state of the suspended particules in theaqueous medium.

When dissolved in isopropyl Myristate the toxicity of the depsipeptideis largely decreased.

What we claim is:
 1. A mixture of peptidolic substances obtained fromthe mycelium of Fusarium equiseti, said mixture being separable intofour components by high pressure liquid chromatography, said componentsbeing designated A, B, C & D, said mixture, on acidic hydrolysisyielding α-hydroxy iso valeric acid and N-methyl Valine; N-methylisoleucine and N-methyl allo iso leucine, and on alkaline hydrolysisyielding the lactone of α hydroxy isovaleroyl N-methyl Valine, thelactone of α-hydroxy isovaleroyl N-methyl isoleucine and the lactone ofα-hydroxy iso valeroyl N-methyl allo iso leucine.
 2. A mixture accordingto claim 1 in which the proportion by weight of the four componentsdesignated A, B, C & D ranges from 2 to 5% for component A, from 10 to16% for component B, from 32 to 38% for component C and from 40 to 47%for compound D.
 3. The component D of the biological substance accordingto claim
 1. 4. The component of claim 3 which upon complete acidhydrolysis yields three moles of a member selected from the groupconsisting of N-methyl isoleucine and N-methyl allo isoleucine per moleof component B and, upon alcaline hydrolysis yields, per mole ofcomponent D, three moles of a lactone selected from the group consistingof lactone of alpha hydroxy isovaleroyl N-methyl isoleucine or alphahydroxy isovaleroyl N-methyl allo iso leucine.
 5. A process forproducing a compound of claim 1 which comprises the steps of aerobicallyfermenting a culture of Fusarium equiseti, separating the mycellium fromthe broth, drying said mycelium and extracting said dried mycelium witha water insoluble organic solvent, separating the organic phase, andremoving the solvent therefrom, to form a residue taking up the residuein a liquid hydrocarbon and exhaustively extracting said hydrocarbonsolution with aqueous alkanol, separating the aqueous alkanol, removingthe solvent therefrom to yield a residue, drying said residue, purifyingsaid residue by chromatography on alumina, concentrating thechromatographic eluate, dissolving said concentrated eluate in analkanol, adding water to said alkanolic solution and removing theprecipitated crystalline compound of claim 1 therefrom.
 6. A processaccording to claim 5 in which the Fusarium equiseti is a mutant speciesdesignated as Fusarium Equiseti (Corda) saccardo filed at TheCommonwealth Mycological Institute at Kew (GB) under the filing number213,107.
 7. A biological substance from the mycelium of Fusariumequiseti 213,107 whenever obtained in or by process of claim
 5. 8. Aprocess of claim 5 comprising the additional purification step ofseparating the products into four components by high pressure liquidchromatography.
 9. The pharmaceutical compositions containing as activeingredient the biological substance of claim 1 in admixture orconjunction with an inert non toxic pharmaceutically-acceptable carrieror vehicle.
 10. The pharmaceutical compositions of claim 9 wherein theinert carriers or vehicles are those suitable for administration by theparenteral the percutaneous, the permucous and the rectal routes.
 11. Apharmaceutical composition according to claim 11 wherein the amount ofactive ingredient ranges from 0.05 to 1 mg per unit dosage.
 12. Apharmaceutical composition containning as active ingredient thebiological substance of claim 10 in admixture or conjunction with aninert non toxic pharmaceutically-acceptable carrier or vehicle.
 13. Amethod for treating or curbing the immunological reactions in the men oranimals suffering from disturbances in the defences of the organism,which consists in administering to said patients a safe but effectiveamount of the biological substance from the Mycelium of a Fusariumaccording to claim
 1. 14. A method of stimulating humoral immunedefenses in subjects suffering from a deficiency of such defenses byadministering to said subjects an effective amount of a substance ofclaim
 1. 15. The method of claim 14 wherein the safe but effectiveamount of the active ingredient ranges from 0.2 to 5 mg per day in theman.
 16. A method of reducing immune reactions in a subject exhibiting ahypersensitivity to same by administering to said subject an effectiveamount of the substance of claim
 1. 17. The method of claim 16 whereinthe safe but effective amount of the active ingredient ranges from 0.2to 5 mg per day in the man.